Process for production of bee venom as pharmaceutical product which can be used effectively in the treatment of rheumatoid arthritis and viral diseases

ABSTRACT

The traditional treatment for Rheumatoid Arthritis relies on a course of synthetic drugs, which range from the use of gold salts to anti-inflammatory drugs including both steroids and non-steroids. These drugs specially the steroids can affect adrenal and pituitary glands &amp; cause impotence, edema, poor wound healing, reduce neurological response and cardiac irregularities.  
     VACSERA began the first clinical studies with bee venom therapy and proved its efficiency for treatment of variety of diseases such as Rheumatoid arthritis and viral infections especially (HCV). Bee Venom was separated by a scientific method and then we determine the dosage form, toxicity, bioavailability, teratogenicity, (anti-teratogenic effect), safety and treatment schedule.  
     The use of Bee venom in treatment of arthritis has been proved to be beneficial to many patients, primarily due to the presence of a number of polypeptides, peptides, enzymes and amines. The venom is administered according to the enclosed leaflet and physician instructions in doses which vary according to the disease and its severity.

BACKGROUND Field of the invention

[0001] The invention relates to the adaptability of the traditionalmedicine (Bee venom) for treatment of a lot of diseases such asrheumatoid arthritis and viral diseases specially (HCV) in apharmaceutical form and presents a noble service for many patients.

[0002] Ancient Egyptians and Babilians were innovators; they used theBee stings to relief pain accompanying Rheumatism and osteoarthritissince 2000 years B.C.

[0003] Chinese have started the bee venom therapy since 1530 In thenineteenth century (1935) French.

[0004] Austrian and Russian doctors began the first clinical studieswith bee venom therapy by using live Bee stings. They proved itsefficiency in treatment of variety of diseases such as Rheumatoidarthritis and viral infections especially (HCV).

SUMMARY

[0005] Nowadays, Apitherapy is practiced by non-professional Bee keepersfor treatment of viral infections (specially HCV), bacterial andrheumatic disease,which leads to many side effects such as cellulitesdue to contamination of the sting pin with microorganism, alsobeekeepers unawareness of a time schedule for the stings may lead tomany serious toxic complications and anaphylactic shock. VACSERA hassucceeded in the separation of the venom by certain electrical device.Then all the pre-clinical studies including the dosage form, treatmentdose, toxicity, bioavaiolability, teratogenicity, and safety wereestablished.

[0006] Then the venom undergoes many purification steps, followed byvenom dilution and sterilization through 0.2 micron depth filter sheet.Afterwards, dispensing of the venom takes place after addition of thepreservative 0.35% Tricresol in vials with known therapeuticconcentration. After that the venom is lyophilized, and is ready for useaccording to the enclosed leaflet and physician instruction in treatmentof viral and rheumatoid arthritis diseases.

DETAILED DESCRIPTION OF THE INVENTION

[0007] Project Goal

[0008] Idealistic use of the venom to modulate both immune cells andimmune mediators in patients suffering from auto-immune diseaseswhenever needed.

[0009] Getting the Bee venom by a sterile and scientific way and inlarge amount, which can

[0010] huge production scale as a pharmaceutical product.

[0011] Manufacturing of the venom locally and no need for importing suchproduct.

[0012] The venom undergoes many purification steps and determination ofits toxicity is established as preliminary procedure before applyingsuch pharmaceutical product for human use either by injection or usingother routes in certain doses according to the severity of the case.

[0013] Then the venom is sterilized by using depth filter sheet withporosity 0.2 micron. Afterwards, packaging of the venom takes placeafter its treatment and mixing with other additives in vials with knowntherapeutic concentration. After that the venom is lyophilized, and isready for use according to the enclosed leaflet and physicianinstructions.

Description in Details of Venom Extraction

[0014] Milking Process:

[0015] Venom of bees was obtained by introducing the bees into certaincabinet of glass lined with

[0016] metal frames, then a small voltage is applied (from 11-15 volts)during the passage of the bees through the metal frames, this processcan stimulate bees to extrude its venom.

[0017] Purification process:

[0018] After drying of the venom, the plates are removed and washedcarefully with sterile physiological saline (NaCl (0.85% )

[0019] Centrifugation in a cold centrifuge for 1 hour must be carriedout at 4500 r.p.m

[0020] Discard the precipitate

[0021] The supernatant, is then lyophilized and kept at 4° C. untilused.

[0022] After lyophilization process, the venom is dissolved in a certainquantity of physiological saline to meet certain concentration/vial

[0023] Adjust PH to 7

[0024] Addition of preservative (tricresol 0.35%)

[0025] Then the solution undergoes sterile filtration through 0.22μdepth filter

[0026] Filling

[0027] Lyophilization

Pharmacological Properties of the Bee Venom

[0028] More than 30 different substances have been characterized in beevenom, the main pharmacological components that reduce inflammation arethe high molecular weight peptides including the following: Mellitin,Apamine, Peptide 401(mast cells degranulating peptide), Adolapin andprotease inhibitors.

[0029] Melittin→Stimulates the hypophyseal adrenal system and releasescortisol that is 100 times more potent than hydrocortisone

[0030] Melittin→Stabilizes the lysosomal cell membrane to protectagainst inflammation and inhibits the complement C₃ system which isinvolved in the inflammatory process

[0031] Ado lapin→Inhibits the microsomal cyclooxygenase system and is 70times stronger than Indomethacine in animal models, it also inhibitsplatelet lipooxygenase, which is involved in the production ofhydroperoxy-icotetranonic acid and leukotrienes. Also it inhibitsthromboxane and prostacycline, which are activated during inflammation.

[0032] Protease inhibitors→inhibit carrageenin, prostaglandin E1,bradykinin and histamine-induced inflammations as well as chymotripsin

[0033] Also bee venom has strong anti-bacterial, antifungal andradio-protective effect by stimulating the heamopoeitic system.

[0034] Bee venom is a strong immunological agent that stimulates thebody protective mechanisms against disease. Chemical properties of beevenom % of Molecular Allergenic Substrate dry venom mass (d) ActivityLow molecular weight <25 <1.000 − Histamine <1 111 − Dopamine <1 153 −Nor epinephrine <1 169 − Amino acids <1 100-200    − Oligo peptides <14200-1.000  − Phospholipids <5 100-400    − Carbohydrates <2 <200 −Peptides <60 <10.000 (+) Melittin <50 2.840 (+) Apamin <2 Tetramer: −12.500 Mast cell degranulating <2 2.000 − peptides (401) Secapin <0.52.600 − tetriapin <0.1 2.000 − protease inhibitor <1 9.000 − Prcomine A& B <2 5.00 − High molecular weight >10.000 (+++) Phosphlipase A <1516-19.000 − Phospholipase B <2 22.000 − Hyaluronidases <2 35-50.000 −Acid phosphomonoesterase <2 45-90.000 (+) D-Glucosidase <1 Not ? known

[0035] Bee venom physical characters

[0036] Single bee venom volume is 5-20 ul

[0037] Venom is colorless, proteinaceous, liquid with sharp bittertaste.

[0038] The dry residue is about 12%

[0039] Has an aromatic odor

[0040] Dried venom has slightly yellowish color

[0041] Determination of LD₅₀

[0042] Once the venom was obtained, determination of the venom lethaldose was carried out as follows:

[0043] Groups of 4 mice (weighing 14-16 gm) were injected in the caudalvein with different concentrations of bee venom

[0044] 1) The first group was injected with 50 ug of bee venom dissolvedin 0.5 ml saline.

[0045] 2) The second group was injected with 75 ug venom in 0.5 mlsaline.

[0046] 3) The third group was injected with 100 ug venom dissolved in0.5 ml saline.

[0047] 4) Finally the 4^(th) group was injected with 150 ug dissolved inthe amount of saline as previously mentioned.

Results

[0048] From the previous work, it is clearly demonstrated that, thelethal dose lies between the 3^(rd) and 4^(th) groups, then additionalinjections with concentrations of 100 ug, 120 ug, and 150 ug (theseconcentrations lie between 100-150ug) take place, all the previousconcentrations caused death in all animal groups. Thus we chose 100 ugas lethal dose which can be identified as the minimum dose of venom thatcauses death in all animal groups within 24 hrs. and the LD₅₀ between70-80 ug

[0049] The lethal dose is 100 ug

[0050] And the LD₅₀ between 70-80 ug

Determination of the Dosage Form

[0051] Our recent work is concerned with the determination of the DOSAGEFORM (either aqueous or haptenuated with complete Freund's adjuvant)ofthe venom, and testing which is better and more useful for immunizationwhen applying such vaccine treatment for human.

[0052] 3 groups of rabbits weighing 3.5-4 kg were injected and immunizedas follows:]

[0053] The first group received 100 ug venom as aqueous preparation,then the schedule was continued for 6 weeks, and before each injection ablood sample is withdrawn to estimate both IgG & IgE levels togetherwith the routine clinical analysis (kidney function, liver function andlipogram tests) to evaluate the effect of the venom on different organsparameters.

[0054] The 2^(nd) group received 100 ug venom emulsified with completefreunds adjuvant, and then as previously mentioned as rabbit No. 1

[0055] The 3^(rd) group received 50 ug emulsified venom to test theeffect of the adjuvant and the extent of amplification of the immuneresponse to bee venom.

Results

[0056] Group No. 1 which is immunized with 100 ug venom as aqueouspreparation, exhibit a gradual increase in the IgG level from 660 mg %to 830 mg %, while IgE level decreased to its minimum level of about 19EU/ml, and no change in the organ functions were observed. (FIGS. 1,2illustrate our results).

[0057] Concerning group No. 2, IgG level increased sharply to a maximumlevel of about 1250 mg %, while IgE level was fluctuating and reached 78EU/ml after the 6^(th) venom injection, also no changes in organfunctions were observed (FIGS. 3,4 reveal that ).

[0058] While the 3^(rd) group which received 50 ug emulsified venomshowed an increase in IgG level to its maximum and reached 1250 mg %,whereas IgE level was fluctuating and reached about 63 EU/ml withoutchange in the organ functions (FIGS. 5,6 explain the previous data).RESULTS were expressed as mean±S.D.

[0059] From the previous work we can depict that the aqueous preparationcan raise the IgG levels gradually and can decrease IgE level to itsminimum level in 2 weeks only, on the contrary although the emulsifiedpreparation induces a higher IgG level, it fails to decrease IgE levelto its minimum value as compared to the aqueous preparation which meansthat, the aqueous preparation can induce complete protection (more thanemulsified preparation) from any deleterious side effects such asanaphylactic shock when applying such treatment for human trails, alsothe addition of complete freund's adjuvant causes some abscesses whenadministered intradermally, due to the composition of the adjuvantitself which contain Tubercle bacilli bacteria that will cause manycomplications for pre-sensitized patients, also hapten-like adjuvant canincrease the incidence of anaphylactic shock.

[0060] Some Facts to be Mentioned

[0061] The aqueous preparation gives complete protection and decreasesthe IgE level during 2 weeks post immunization with sustained increasein the IgG level.

[0062] Although the administration of the dose as emulsified preparationcauses higher increase in the IgG level, we must avoid that form ofinjection due to the elevation of the IgE level and the abscesses formedduring intradermal injection.

Toxicity

[0063] Effect of the Venom on the Renal System

[0064] Female Wister rats weighing 150-200 gm were injectedintravenously with Africanized bee venom at a dose of 0.4 ul/100 gm ofbody weight and used in functional and light microscopy studies. Theanimals were divided into 2 groups: the early group was studied 3-8 hourafter inoculation, and the late group was studied 24-30 hoursthereafter. The animals showed acute renal failure characterized byreduction of glomerular filtration rate with elevation of plasmacreatinine. They also showed increased fractional sodium and potassiumexcretions, suggesting changes in the proximal portion of the nephron.The water transport through collecting tubules was reduced, withconsequent diuresis, indicating functional changes in the distal portionof the nephron. These functional changes were more marked in the earlygroup, with recovery tending to occur after 24 hr, albuminuria was alsoobserved in this group. Light microscopy showed acute tubular necrosismainly in cortex and outer medulla, with isolated necrosis in cells orsmall groups of cells and cast formation in the distal and collectingtubules. After 24 hour frequent mitotic figures were found in thetubular epithelium.

[0065] The observed acute renal failure was due to acute tubularnecrosis which in turn was properly caused by multiple effects, mainlyhemodynamic changes secondary to cardio-toxicity and systemicvasodilatation caused by the venom, myohemoglobinuria and the directaction of the venom on tubular cells.

[0066] Effect on Cardio-Vascular System

[0067] An infarct like myocardial lesions was observed in Wister ratsafter inoculation of high amount of the venom intravenously.

Evaluation of Mutagenicity

[0068] The mutagenic effect of bee venom was assessed bysalmonella/microsome, the venom exerts an antimutagenic effect againstthe mutagenicity of 4-nitro-phenylenediamine and daunomycin.

[0069] Bee venom has an excellent tolerability and wide safety margin upto 700 μg/kg of body weight

[0070] Immunotherapy with Bee venom leads to complete protection in morethan 98%of the patients with a history of hyperallergy to the venom.

[0071] At much higher doses anaphylaxis, pruritis, nettle rash,myxoedema, spasms of the smooth muscles and sudden decrease of bloodpressure may develop.

[0072] Histamine content of Bee venom at a high dose may cause spasms ofcoronary vessels

[0073] But itching usually shows future good therapeutic results.

[0074] Bee venom therapy also leads to increase masculinedifferentiation.

[0075] Immune-therapy during pregnancy did not lead to allergicsensitization of patients' children.

Bee Venom Pharmacokinetics

[0076] Concerning the information that have been requested regarding thepharmacokinetics of bee venom.

[0077] First this point is still under research and inexplicit due tomultiple components included in the venom (many peptides, polypeptides,enzymes and amino acids ),the molecular weight of the components rangingfrom 1000 to 90,000 Da, which makes the study lasts more time. Howeverour preliminary data (animal model) elicits that the major allergencomponent of the venom is Phospholipase A2 and melittin, also thepreviously mentioned peptides are the most common peptides which inducepharmacological effects together with the Apamin (12,500 Da) of the beevenom, venom fractions of lower molecular weight were pharmacologicallyinactive.

[0078] Nevertheless, toxicokinetics of both classes of venom componentswere studied

[0079] After venom intravenous injection with a dose of( 70 ug/kg) thevenom plasma level followed a bi-exponetial decline with distributionhalf life of 45 min. and an elimination half life of 1.8 hr and thesystemic clearance 60 ml/h/kg

[0080] Venom level in plasma, after S.C. injection of a dose (700ug/kg)of venom, increased within a few hours after venom administrationto reach a maximum value at 5±0.5 hr. They subsequently followed amonoexponential decline.

Evaluation of Excretion Route

[0081] The route of excretion is determined using intact andnephrectomized rats, after injection of bee venom, the initial 15 min.of the half life was considerably longer in nephrectomized animals afterinjection, so we concluded that the proximal tubules cells of the kidneyparticipate in the metabolism of circulating venom and higher venomlevels persist in plasma of nephrectomized animals. For that reason theimmunotherapy with Bee venom is restricted for patients with renalimpairment.

Evaluation of Anti-Bacterial Activity

[0082] Also the evaluation of anti-bacterial activity of the bee venomwas established by Melittin, the mechanism by which it exerts itsaction, is not yet clarified but it may be due to the formation ofpeptide-lipid supramolecular complex pore in the membrane, followed bypeptide internalization, simultaneously dissipating the trans-membranepotential and the lipid asymmetry, this also would be of value indeveloping a more potent antibiotic based on these results.

[0083] All the previously mentioned data is due to preliminary studyonly, and after the completion of this point we will send all thedetails in. CLINICAL TRIALS a) For rheumatoid Arth. Patients

b) For Viral infected patients (HCV infected patients)

[0084] Before application of such therapy, patient must fulfill thefollowing record.

[0085] Firstly patient must sign consent form including his agreement toundergo such trials

[0086] Personal sheet includes

[0087] Sex

[0088] Name

[0089] Date of birth

[0090] Marital status

[0091] Address

[0092] Phone

[0093] Present occupation

[0094] Previous occupation

[0095] Main Complaint

[0096] History of the Previous Illness

[0097] Chronic Illnesses Associated

[0098] Hypertension

[0099] D.M.

[0100] Cardiac

[0101] Chest

[0102] Renal

[0103] Liver

[0104] Bilharzias

[0105] Other

[0106] Drug history

[0107] Previous occupation

[0108] Previous blood transfusion

[0109] Investigation Done

[0110] Past History

[0111] Family History

[0112] Medical Examination Report

[0113] General examination

[0114] Blood pressure

[0115] Pulse

[0116] Temp.

[0117] Appearance

[0118] Head &neck

[0119] Chest

[0120] Abdomen

[0121] Limbs

[0122] Local Examination

[0123] Professional Diagnosis

[0124] Investigations Required

[0125] Laboratory

[0126] Radiology

[0127] Others

[0128] Diagnosis

[0129] Recommendations

[0130] Treatment Schedule

[0131] Rush schedule

[0132] Recommended maintenance schedule (including time interval betweeninjections)

[0133] Special Injection Schedule (due to Bernstein et al 1993,1994 andDiaz Gomez et al 1995) was taken and modified according to the type ofdisease and its severity. This schedule is in conformance with thescientific literature recommendations, which have been published in thisfield. (Bernstein et al 1993,1994 and Diaz Gomez et al 1995)

[0134] Base line analysis included

[0135] Liver function tests,

[0136] Kidney function tests,

[0137] lipogram, and

[0138] Fasting blood sugar

[0139] to detect the effect of the BEE VENOM on them later on.( for anyevidence of toxicity).

Vacsera Schedule can be Summarized as Follow

[0140] Rush Immunization Week

[0141] Day 1:

[0142] Patient is injected with 25u of the venom solution s.c or i.d.divided on both hands.

[0143] Day 3:

[0144] Patient is injected with 50 u of the venom solution s.c or i.d.divided on both hands.

[0145] Day5:

[0146] Patient is injected with 75u of the venom solution s.c or i.d.divided on both hands.

[0147] Day 7:

[0148] Patient is injected with 100 u of the venom solution s.c or i.d.divided on both hands.

[0149] After 2 weeks:

[0150] Patient is injected with another 100 u of the venom solution s.cor i.d. but undivided.

[0151] Maintenance Treatment

[0152] i) For Rheumatic and HCV infected patients

[0153] A dose of 100-200 ug can be administered twice weekly for 6 weeks

[0154] ii) For hyper-allergic patients against bee venom

[0155] Patient is injected monthly with 100 u of the venom for 8 months

[0156] Recommendations

[0157] Patients having high IgE level must be treated carefully andappropriate precautions are taken.

[0158] Analysis recommended for every case is repeated every 21 days tocheck any evidence of toxicity.

[0159] HCV infected Patients, detection of virus removal by PCR arecarried out every month to check the rate of viral eradication togetherwith liver function tests.

[0160] Rheumatoid arthritis patients are checked permanently for

[0161] ESR,

[0162] RF-latex

[0163] Anti-DNA for sero-negative rheumatoid patients,

[0164] ANA,

[0165] Interleukin 1 and Interleukin2

[0166] besides complete check up for any evidence of toxicity.

[0167] Asthmatic patients due to elevation of IgE titer are checked forIgG and IgE every 2 weeks.

[0168] Precautions

[0169] Divided doses should have time interval 30 min.

[0170] Patients with high IgE level should be given Corticosteroids andantiallergic such as Fexofenadin Hcl 180 mg and calcium salts during thedays of immunization.

[0171] During the rush immunization week, patient stays underobservation for 2 hours for any expected deleterious side effects.

[0172] Avoid intravenous injection of the venom with huge amount thatmay lead to infarction like lesions (Animal model trial).

[0173] It has no mutagenic effect and does not cross placental barrier

[0174] Intravenous injection of the venom can lead to destruction ofkidney microtubules.

[0175] Applications

[0176] Used by individuals, clinics and hospitals as a pharmaceuticalbiological product

[0177] either inject able according to certain immunization schedules tomodulate

[0178] immune cells and immune mediators and eradicates viral infection

[0179] Or in other forms like cream, lotion, and plasters to treat a lotof diseases.

We claim 1- The venom extraction method (milking process). 2- Method ofclaim 1, in which the bees undergo milking process several times with nodecrease in bees number. 3- The method of purification, by which thevenom is obtained in the most pure form. 4- The therapeutic benefitswhich have been established for every fraction of the 33 fractions ofthe venom. 5- According to claim 4,where in the Melittin can achieve itstherapeutic effect by stimulation of hypophyseal-adrenal gland torelease cortisol and stabilizes the lysosomal cell membrane againstinflammation and inhibits the complement C3 system that is involved inthe inflammation process. 6- According to claim 4,where, Adolapininhibits microsomal cyclooxygenase system and also inhibits thromboxaneand prostacycline, which are activated during inflammation. 7- Accordingto claim 4,where, protease inhibitors inhibit carrageenin, prostaglandinE₁, bradykinin and histamine induced inflammation as well aschymotripsin. 8- The administration method including both rush andmaintenance treatment. 9- A process according to claim 8, where the weekof rush injection is recommended for patient desensitization against anyfuture adverse effects, in which ascending doses are administered from25 to 100 μg. 10- A process according to claim 8, including maintenancedose of about 100-200 μg can be administered twice weekly for rheumatoidarthritis & viral infected patients. 11- A process according to claim 8,in which patients who exhibit hypersensitivity against bee venom aredesensitized by using 100 μg of the venom for 8 months as monthlyinjected dose 12- A process of claim 8,in which the duration of thetreatment is not less than 8 consecutive weeks. 13- The recommendeddosage form of the venom, whereas the administration of aqueouspreparation raises IgG level gradually and lowers IgE level to itsminimum value in two weeks only, reveals dual benefits in preventing themain cause of anaphylactic shock during the maintenance treatment andavoiding sudden elevation of IgG against the venom due to the immunetolerance that originated as a result of administration of allergen inhigh doses with narrow time intervals.